Download BD™ Brucella Blood Agar with Hemin and Vitamin K1

INSTRUCTIONS FOR USE –
READY-TO-USE PLATED
MEDIA
PA-255509.05
Rev.: Sep 2011
BD Brucella Blood Agar with Hemin and Vitamin K1
INTENDED USE
BD Brucella Blood Agar with Hemin and Vitamin K1 is a highly nutritious medium for the
isolation and cultivation of strict anaerobes from clinical specimens.
PRINCIPLES AND EXPLANATION OF THE PROCEDURE
Microbiological method.
BD Brucella Blood Agar with Hemin and Vitamin K1 is a modification of Brucella Agar that
has been supplemented with hemin and vitamin K1 to support the growth of fastidious
anaerobes, especially Bacteroides, Prevotella, and Porphyromonas when incubated
anaerobically.1-3 It is used for the isolation of strict anaerobes from clinical specimens. It is also
used for susceptibility testing of anaerobes with the E test method.4-6
In BD Brucella Blood Agar with Hemin and Vitamin K1, peptones and yeast extract, together
with glucose, supply nutrients. Sodium bisulfite lowers the redox potential to a range suitable for
strict anaerobes. Hemin and vitamin K1 have been shown to be necessary for supporting the
growth of certain strict anaerobes.7 Sheep blood provides additional nutrients and is used to
detect hemolytic reactions.
REAGENTS
BD Brucella Blood Agar with Hemin and Vitamin K1
Formula* Per Liter Purified Water
Pancreatic Digest of Casein
10.0 g
Peptic Digest of Animal Tissue
10.0
Yeast Extract
2.0
Glucose
1.0
Sodium Chloride
5.0
Sodium Bisulfite
0.1
Hemin
0.005
Vitamin K1
0.01
Agar
15.0
Sheep Blood, defibrinated
5%
pH 7.2 +/- 0.2
*Adjusted and/or supplemented as required to meet performance criteria.
PRECAUTIONS
. For professional use only.
Do not use plates if they show evidence of microbial contamination, discoloration, drying,
cracking or other signs of deterioration.
Consult GENERAL INSTRUCTIONS FOR USE document for aseptic handling procedures,
biohazards, and disposal of used product.
STORAGE AND SHELF LIFE
On receipt, store plates in the dark at 2 to 8° C, in their original sleeve wrapping until just prior to
use. Avoid freezing and overheating. The plates may be inoculated up to the expiration date
(see package label) and incubated for the recommended incubation times.
Plates from opened stacks of 10 plates can be used for one week when stored in a clean area
at 2 to 8° C.
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USER QUALITY CONTROL
Inoculate representative samples with the following strains (for details, see GENERAL
INSTRUCTIONS FOR USE document). Incubate for 48 to 72 hours in an anaerobic atmosphere
(e.g. BD GasPak Anaerobic System) at 35 to 37° C.
Strains
Bacteroides fragilis ATCC 25285
Clostridium perfringens ATCC 13124
Growth Results
Growth good to excellent; grey colonies
Growth good to excellent; large, lobate, greywhite colonies; beta (double-zone) hemolysis
Growth good to excellent; grey-white colonies,
surrounded by dark-grey zones
Growth good to excellent; whitish colonies
Fusobacterium nucleatum ATCC 25586
Peptostreptococcus anaerobius
ATCC 27337
Porphyromonas levii ATCC 29147
Growth fair to good; dirty whitish to grey-brown
colonies
Red to dark red (blood color)
Uninoculated
PROCEDURE
Materials Provided
BD Brucella Blood Agar with Hemin and Vitamin K1 (90 mm Stacker plates).
Microbiologically controlled.
Materials Not Provided
Ancillary culture media, reagents and laboratory equipment as required.
Specimen Types
BD Brucella Blood Agar with Hemin and Vitamin K1 is a universal medium for the isolation
and cultivation of strict anaerobes from all types of specimens (see also PERFORMANCE
CHARACTERISTICS AND LIMITATIONS OF THE PROCEDURE). Observe approved
techniques for collection and transport of anaerobic specimens.8-10 Suitable transport media,
e.g., BD Port-A-Cul, must be used.
BD Brucella Blood Agar with Hemin and Vitamin K1 is also used for susceptibility testing of
strict anaerobes with the E test which requires the use of pure cultures.4-6
Test Procedure
Streak the specimen directly after arrival, using an approved streaking technique onto BD
Brucella Blood Agar with Hemin and Vitamin K1. Directly after streaking, place the plates into
anaerobic jars supplied with an anaerobic atmosphere. It is recommended to use BD GasPak
jars and BD GasPak H 2 /CO 2 envelopes together with a catalyst. Incubate for 2 to 3 days or
longer, if necessary, at 35 to 37° C. Regardless of the anaerobic system used, it is important to
include an indicator of anaerobiosis such as the BD GasPak disposable anaerobic indicator.
If specimens containing mixed flora are streaked onto the medium, it is recommended to also
include selective media such as BD Schaedler Kanamycin-Vancomycin Agar with 5% Sheep
Blood and/or BD Wilkins-Chalgren Agar with Amikacin and 7% Sheep Blood. Also,
facultative anaerobes might be present in the specimen. Therefore, it is recommended to always
include an aerobic medium (such as BD Columbia Agar with 5% Sheep Blood) when the
primary cultures are set up. This plate is incubated aerobically enriched with carbon dioxide
together with the anaerobic cultures.8 It allows the detection of facultative organisms in the
specimen.
For use of BD Brucella Blood Agar with Hemin and Vitamin K1 in anaerobic susceptibility
testing with the E test, consult the references or the instructions of the manufacturer.4-6
Results
After incubation, the plates are inspected for growth. Colonies which appear on this medium
are suspected to be strict anaerobes if they do not grow on aerobically incubated blood agar
plates. Also, growth on BD Schaedler Kanamycin-Vancomycin Agar with 5% Sheep Blood
is compared to the growth on the other media. If mixed cultures of strict and facultative
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anaerobes are present, appropriate subcultures on non-selective media, incubated aerobically
and anaerobically, must be made from the anaerobic media to confirm that the isolate is a strict
anaerobe.
Further microscopic and biochemical examination is necessary for the identification of the
genera and species of the strict anaerobes. Consult appropriate texts for further information,
including identification procedures.6, 8,9,11
For reading the E test results obtained on this medium, consult the references or the instructions
of the manufacturer. 4-6
PERFORMANCE CHARACTERISTICS AND LIMITATIONS OF THE PROCEDURE
On BD Brucella Blood Agar with Hemin and Vitamin K1 which is one of the nonselective
standard media for the isolation of strict anaerobes, Bacteroides, Prevotella, Porphyromonas,
Fusobacterium, Clostridium, Peptostreptococcus, strictly anaerobic non-sporeforming rods
(e.g., the former genus Eubacterium), Mobiluncus, Actinomyces and many others will grow.6,8-11
Additionally, it is used for susceptibility testing with the E test method.4-6
Performance Results12
BD Brucella Blood Agar with Hemin and Vitamin K1 was evaluated internally with clinical
isolates and collection strains of the following strictly anaerobic species and compared to BD
Schaedler Agar with Vitamin K1 and 5% Sheep Blood (= reference medium): Bacteroides
fragilis, B. distasonis, Prevotella bivia, Fusobacterium nucleatum, Porphyromonas levii,
Porphyromonas gingivalis, Campylobacter (Bacteroides) gracilis, Peptostreptococcus
anaerobius, Clostridium perfringens, Mobiluncus mulieris, Eggerthella lenta (Eubacterium
lentum). Both media were inoculated with 103 to104 cfu per plate and were incubated
anaerobically for 3 days . The number of colonies on the test medium was equal to or higher
than that on test reference medium for all of the strains tested.
Limitations of the Procedure
Note that the growth rates of strict anaerobes vary considerably: While Bacteroides fragilis will
grow well after 24 hours, Mobiluncus or strains of Porphyromonas need 4 to 5 days, and
Actinomyces may need 1 to 3 weeks to produce well visible colonies. If cultures are negative
after 2 or 3 days of incubation, re-incubate anaerobically for an additional 2 to 3 days. If
Actinomyces is suspected, dedicated culture plates of this and other media (e.g., BD
Columbia Agar with 5% Sheep Blood) should be inoculated that are inspected after 1, 2 and
eventually 3 weeks of incubation.
This medium is not specifically selective for strict anaerobes; facultative organisms will also
grow. Therefore, it is important to compare the result of the anaerobic culture with that of an
aerobically incubated plate if mixed cultures are obtained.
REFERENCES
1. MacFaddin, J.F. 1985. Media for isolation cultivation-identification-maintenance of medical
bacteria, vol. I. Williams & Wilkins, Baltimore.
2. Gibbons, R.J., and J.B. MacDonald. 1960. Hemin and vitamin K compounds as required
factors for the cultivation of certain strains of Bacteroides melaninogenicus. J. Bacteriol.
80:164-170.
3. Finegold, S.M., V.L. Sutter, H.R. Attebery, and J.E. Rosenblatt. 1974. Isolation of anaerobic
bacteria, p. 368. In E.H. Lennette, E.H. Spaulding, and J.P. Truant (ed.), Manual of clinical
microbiology, 2nd ed. American Society for Microbiology, Washington, D.C.
4. Citron, D.M., et al. 1991. Evaluation of the E test for susceptibility testing of anaerobic
bacteria. J. Clin. Microbiol. 29: 2197-2203.
5. Church, D.L., et al. 1996. Metronidazole susceptibility and the presence of hydrogenase in
pathogenic bacteria.
6. Citron, D.A., and D.W. Hecht. 2003. Susceptibility test methods: anaerobic bacteria. In:
Murray, P. R., E. J. Baron, J.H. Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.). Manual of
clinical microbiology, 8thed. American Society for Microbiology, Washington, D.C.
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7. Gibbons, R.J., and J.B. MacDonald. 1960. Hemin and vitamin K compounds as required
factors for the cultivation of certain strains of Bacteroides melaninogenicus. J. Bacteriol.
80:164-170.
8. Engelkirk, P.G., J. Duben-Engelkirk, and V.R. Dowell, Jr. 1992. Principles and practice of
clinical anaerobic bacteriology. Star Publishing Co., Belmont, Calif.
9. Summanen, P., E.J. Baron, D.M. Citron, C.A. Strong, H.M. Wexler, and S.M. Finegold.
1993. Wadsworth anaerobic bacteriology manual, 5th ed. Star Publishing Co., Belmont,
Calif.
10. Murray, P.R., and D.M. Citron. 1991. General processing of specimens for anaerobic
bacteria. In: A. Balows, W.J. Hausler, Jr., K.L. Herrmann, H.D. Isenberg, and H.J. Shadomy
(ed.), Manual of clinical microbiology, 5th ed. American Society for Microbiology,
Washington, D.C.
11. Murray, P.R., E.J. Baron, M.A. Pfaller, F.C. Tenover, and R.H. Yolken (ed.). 1995. Manual
of clinical microbiology, 6th ed. American Society for Microbiology, Washington, D.C.
12. Data on file. BD Diagnostic Systems Europe. Heidelberg, Germany.
PACKAGING/AVAILABILITY
BD Brucella Blood Agar With Hemin And Vitamin K1
Cat. No. 255509
Ready-to-use Plated Media, cpu 20
FURTHER INFORMATION
For further information please contact your local BD representative.
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Tullastrasse 8 – 12
D-69126 Heidelberg/Germany
Phone: +49-62 21-30 50
Fax: +49-62 21-30 52 16
[email protected]
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